Journal
PROTEOMICS
Volume 10, Issue 20, Pages 3644-3656Publisher
WILEY
DOI: 10.1002/pmic.201000190
Keywords
FT-MS; Membrane proteins; Photosystem II; Red algae; Technology; Top-down proteomics
Funding
- NIH [S10 RR023045]
- National Science Foundation NSF [0417142]
- DFG [SFB TR1]
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High-resolution top-down MS was used to characterize eleven integral and five peripheral subunits of the 750 kDa photosystem II complex from the eukaryotic red alga, Galdieria sulphuraria. The primary separation used LC MS with concomitant fraction collection (LC-MS+), yielding around 40 intact mass tags at 100 ppm mass accuracy on a low-resolution ESI mass spectrometer, whose retention and mass were used to guide subsequent high-resolution top-down nano-electrospray FT ion-cyclotron resonance MS experiments (FT-MS). Both collisionally activated and electron capture dissociation were used to confirm the presence of eleven small subunits to mass accuracy within 5 ppm; PsbE, PsbF, PsbH, PsbI, PsbJ, PsbK, PsbL, PsbM, PsbT, PsbX and PsbZ. All subunits showed covalent modifications that fall into three classes including retention of initiating formyl-methionine, removal of methionine at the N-terminus with or without acetylation, and removal of a longer N-terminal peptide. Peripheral subunits identified by top-down analysis included oxygen-evolving complex subunits PsbO, PsbU, PsbV, as well as Psb28 (PsbW) and Psb27 (PsbZ-like). Top-down high-resolution MS provides the necessary precision, typically less than 5 ppm, for identification and characterization of polypeptide composition of these important membrane protein complexes.
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